The present invention relates to an apparatus and a method for measuring the optical properties of a medium, and a corresponding micro-bioreactor. These optical properties may in particular include the turbidity, the absorbance and the fluorescence of the medium, at one or more wavelengths or over predetermined spectral ranges.
In many biological laboratories and also in certain industries, it is very often necessary to cultivate micro-organisms in liquid medium and to measure the concentration of the cells at different stages of the culture. This quantification may be performed directly on samples, by counting the cells or by measurement of the dry weight. These methods, although very accurate, are long and tedious, and it is more convenient to evaluate the quantity of the cells indirectly, for example by measuring the disappearance of substrates or the appearance of intracellular (NADH, etc.), or extracellular compounds (alcohols, organic acids, etc.). However, the most convenient and most frequently used methods for evaluating the cell concentrations of cultures are based on their optical properties.
When matter is struck by electromagnetic radiation, this interacts with the electronic charges of the atoms: part of the radiation passes through the matter without change of direction (transmitted radiation) and part is diffused in all directions, each illuminated particle then behaving as a light source. The quantity of light diffused by the particles present in the liquid medium increases with the number and size of these particles; in addition, in the case where the particles are micro-organisms, it has been shown that the diffusion of the light does not occur homogeneously in all directions, but predominantly in a direction close to that of the incident beam.
It is these diffusion phenomena which cause liquid micro-organism cultures to have a cloudy appearance, whose density increases with the cell concentration. Devices which can quantify this cloudiness, or turbidimeters, measure the turbidity of these cultures.
Existing turbidimeters may be divided into discontinuous measurement devices, which are used to make repeated measurements of the light transmitted and/or reflected by a liquid medium, and on-line measurement devices, for which a turbidity measurement probe is inserted into the liquid medium and connected to a recording system.
Discontinuous measurement devices, such as nephelometers, colorimeters, spectrophotometers and mixed turbidimeters, cannot for the most part measure high turbidity levels. Their measurement range does not normally extend beyond 1000 NTU (Nephelometric Turbidity Units) and is most often much more limited. It is thus necessary to dilute the samples, which increases the risks of error and the work to be performed by the operator. In addition, the sampling itself is very restricting, since it requires the presence of the operator at regular intervals throughout the culture period. Some mixed turbidimeters have been designed to cover a wider turbidity range, but they are costly.
On-line turbidimeters, whose measurement range may be fairly wide, have the disadvantage of requiring large culture volumes (about 1 litre) because of the size of the probes introduced into the cultures, which limits their use within bioreactors. In addition, cultures in bioreactors are generally aerated and stirred, which generates numerous bubbles which interfere strongly with turbidity measurement. These devices are also costly.
International patent application WO-92/13.482 discloses an apparatus and a method for measuring a blood parameter. The apparatus comprises a red light source and an infrared light source directing the light towards a blood sample and a detector receiving the light generated by the two sources and reflected by the blood. An optical feedback loop is used for each of the two sources so that the intensity of the light received by the detector is approximately constant for a range of values of the blood parameter, which allows precise control of the light sources. Reference curves are used to calculate two blood parameters from the feedback signals obtained respectively for each of the two sources.
A disadvantage of this technique is that it is in particular limited by the transmission capacities of the light sources and thus only covers a restricted measurement range with sufficient sensitivity. In addition, obtaining each result requires a stabilisation time which prevents rapid measurement. A serial analysis of a large number of samples, or measurement in heterogeneous media and /or media containing gas bubbles thus proves very difficult, or even impossible. In addition, inaccuracies can be generated during the processing of the feedback signals, because of the drift of capacitance values.
U.S. Pat. No. 4.447.150 relates to a device and a method for measuring blood properties and parameters, used to measure the oxygen saturation level of the blood. The apparatus disclosed comprises two light sources transmitting respectively at two distinct wavelengths and a detector measuring the light generated by the two sources and reflected by the blood. The light reflected or transmitted by the blood is maintained at a constant level by means of an optical feedback loop. In addition, the ratio of the two voltages corresponding to the measured light levels from the two sources respectively gives the percentage of saturation of the blood oxygen.
This technique also has the disadvantage of only allowing a restricted measurement range, in addition to the disadvantages already mentioned for document WO-92/13.482.
The present invention provides a measurement apparatus and a method which do not have the above disadvantages, and thus allow measurements over different ranges of values while retaining good sensitivity.
The apparatus and method of the invention can also offer a high speed of measurement and good accuracy.
In particular, the object of the invention is an apparatus for measuring turbidity able accurately to measure the turbidity of microbial cultures over a wide range of values, which is reliable, easy to use, readily adaptable to micro-bioreactors, of small size and inexpensive.
A further object of the invention is an apparatus for measuring absorbance and an apparatus for measuring fluorescence, able to measure with the same advantages as cited above the molecular absorbance and/or fluorescence of a medium, and, more generally, the invention provides an apparatus for measuring one or more optical properties of a liquid or solid medium.
The invention relates to a measurement apparatus as described above which is advantageously automated, so that experiments may be performed over a long time period without the presence of an operator.
Another object of the invention is a measurement apparatus as described above suitable for a battery of micro-bioreactors, making simultaneous measurements for the different micro-bioreactors possible.
The invention also relates to a micro-bioreactor equipped with a measurement apparatus having the above properties.
A particular object of the invention is a micro-bioreactor having systems for supplying the culture medium and gas which are simple and reliable, protected from any external contamination and able to handle small culture volumes. The system similarly protects the external environment from contamination by the culture itself.
The invention also relates to a micro-bioreactor with automatic sampling at certain predetermined stages of turbidity, absorbance, and/or fluorescence, and a micro-bioreactor with automatic dilution for cyclic cultures.
A further object of the invention is a method for measuring optical properties which may include in particular turbidity, absorbance and fluorescence, making possible measurement over a wide range of values with good accuracy, reliability and reproducibility.
The invention may be applied in the fields of microbiology and biotechnology, and more generally for any measurement of turbid substances, for example for analysis and treatment of water, agricultural or industrial effluents or pollutants (turbidity measurement).
It may be also be applied to in vivo or in vitro measurement of fluorescence, and more precisely to measurement of the concentration of intra- or extracellular fluorescent compounds.
The object of the invention is thus an apparatus for measuring at least one optical property of a medium, comprising:
a light source which transmits a light beam of variable intensity towards and into the medium,
detection means to measure the intensity of the beam modified by the liquid medium,
means of controlling the intensity of the beam transmitted by the source,
storage means which store a representative value of a nominal intensity of the modified beam,
a feedback system linked to the control means, the detection means and the storage means, operating on the control means so that the measured intensity of the modified beam is equal to the nominal intensity, and
means of reading the intensity of the transmitted beam, for the nominal intensity of the reflected beam, the intensity of the transmitted beam and the nominal intensity of the modified beam being representative of the optical property or properties of the medium.
According to the invention, the measurement apparatus comprises means for adjusting the nominal intensity, linked to the reading means and the storage means, designed to adjust the nominal intensity so that the stabilised intensity of the transmitted beam falls within a predetermined range.
The nominal intensity of the modified beam is adjustable . It is thus possible to adapt the measurement apparatus to a given range of turbidity, absorbance and fluorescence values. The choice of the nominal intensity results from a compromise. It must be as high as possible, so that the intensity of the transmitted beam varies strongly with the turbidity, absorbance and/or fluorescence, which gives high accuracy. However, the nominal intensity must also be sufficiently reliable to cover the whole of the desired range without the intensity of the transmitted beam exceeding a maximum transmission threshold of the source, causing its saturation and/or deterioration.
This apparatus may thus be used for measurements of different ranges of values, from very low to very high levels, while retaining optimal sensitivity. In contrast, in the prior art the measurement range is fixed and linked to the reference voltage used.
In particular, the apparatus makes turbidity measurements possible over a wide range of optical densities with good accuracy. It is thus possible to obtain without difficulty a range extending from 45 to more than 17 000 NTU, which for a suspension of Saccharomyces cerevisiae yeast corresponds respectively to from 0.2 to 75 units of optical density (OD) at 600 nm with an error of about 2%. This apparatus also has good reliability and good reproducibility of measurements, corresponding to a low dispersion of values. Correspondence curves between turbidities and cell concentrations may easily be constructed for different organisms.
In addition, the measurement apparatus according to the invention may be manufactured at low cost, since it makes use of a relatively small number of inexpensive electronic components.
The storage means are advantageously designed to store a saturation intensity and the adjustment means regulate the nominal intensity to a lower value when the stabilised intensity of the transmitted beam is higher than the saturation intensity.
The values taken for the nominal intensity are advantageously predetermined.
The adjustment of the nominal intensity is thus automated, which in particular allows the developing turbidity of a medium to be monitored, over a wide turbidity range and with very good accuracy. It is particularly advantageous to use a luminescent diode, preferably transmitting in the infrared around 880 nm, for this automated adjustment. The voltage applied to the terminals of the transmitting diode during the transmission of the light beam in fact increases proportionately with the turbidity, thus giving excellent measurement accuracy.
The medium studied may be liquid or solid (gels, microscopy preparations).
The optical property or properties measured are advantageously selected from amongst the turbidity T, the absorbance A or the fluorescence F of the medium, for a given couple of transmission and reception spectra. Different embodiments of the invention include the following different combinations of the types of transmitted and received light:
monochromatic or wider band light source, detection at a corresponding wavelength (monochromatic or wider band),
monochromatic or wider band light source, detection at another wavelength (case of fluorescence),
polychromatic or wider band light source, detection at several corresponding wavelengths,
white light source, detection at one or more wavelengths selected by filters.
The detection of the intensity of the beam received at several wavelengths can give additional information on the medium.
The light source is composed of a transmitter such as for example a diode, laser, coupled or not with an optical fibre, designed to transmit monochromatic light, light at spectral bands, possibly with variable widths, or white light. The transmission may be in the UV, visible, or near or far-infrared regions.
The detection means advantageously consist of one or more photodetectors, such as for example diodes (phototransistor, photodarlington) or photoelectric cells, each having monochromatic or a wider passband reception, in the UV, visible, or near or far-infrared regions. In the case of infrared measurements, the detection means are advantageously insensitive to visible light, for convenience.
The language xe2x80x9cbeam modified by the medium,xe2x80x9d and similar terminology appearing in the specification and claims should be understood to mean a beam transmitted through the medium without interaction, a diffused or reflected portion of a beam entering the medium, or a beam transmitted after interaction with the medium (in the case of fluorescence, for example).
The detection means are placed at one or more appropriate angles with respect to the direction of the transmitted beam, preferably at about 180xc2x0 (necessarily at about 180xc2x0 for transmission).
The beam incident on the medium is preferably parallel. This gives a very good ratio of modified light to transmitted light.
Each wavelength measured is preferably selected so as to give information on a distinct type of particle.
Thus, for turbidity measurements, two distinct wavelengths lead to results on particles of two different sizes coexisting in suspension. These might for example be cells infected by phage particles and phages liberated by the lysed cells. For a given type of particle, the quantity of light diffused or transmitted depends on the wavelength detected. In addition, the higher the wavelength, the higher the transmitted light/transmitted light ratio.
An embodiment for measurement at two wavelengths consists of transmitting using two transmitters of different wavelengths, for example infrared and visible. Another embodiment consists of transmitting white light and placing filters on the detector so as to select the two wavelengths.
For absorbance measurements, reception of two distinct wavelengths allows quantification of two types of molecules absorbing at two distinct wavelengths, or the correlation of observations on a type of particle.
In general, measurements at several wavelengths can be used to determine the proportion and nature of the particles, cells or molecules in the media analysed, and to distinguish their size, shape, concentration, nature and/or the proportion of different cell, particle or molecular species. If the number of wavelengths is equal to n, a mathematical process may be performed using a system of n equations with n unknowns.
The nominal intensity (fixed intensity of the modified beam) is advantageously selected as a function of the range of concentration of the particles to be measured in the medium, the nominal intensity decreasing when the concentration range to be measured increases.
The measurement apparatus according to the invention does not simply measure the light modified by a liquid medium to determine the turbidity, absorbance, or fluorescence, but the energy supplied by the source in order to obtain a fixed quantity of modified light. This energy, or intensity of the transmitted beam, gives the turbidity, the absorbance or the fluorescence of the liquid or solid medium, for each wavelength measured, by means of a simple mathematical relation established by calibration curves. Thus, when the light source transmits an infrared light around 880 nm, the quantity of energy transmitted by the medium is inversely proportional to the turbidity of the medium passed through and the electronic feedback system automatically adjusts the transmitted beam so that the modified light is equal to a fixed value.
In the case of fluorescence measurement, the quantity of light modified is proportional to the intensity of the transmitted beam and to the concentration of the fluorescent substance. It is also affected by internal filter phenomena.
The measurement apparatus also obviates any removal of portions of the medium other than for analysis samples, which simplifies handling operations, avoids error risks during dilution after sampling and reduces the risks of contamination of the culture or the environment (for example in the case of pathogen cultures).
The measurement apparatus may advantageously be completely outside the liquid medium. Thus, in the case where the medium comprises a culture, the apparatus does not have to be sterilised and is not liable to deterioration by fouling or coating with biofilms.
The measurement apparatus is preferably automated, in particular by automatic recording of data and by automatic adjustment of the nominal intensity of the modified beam. It is thus possible to perform long experiments without the presence of an operator, in particular for monitoring cultures on a 24-hour basis without human supervision.
Another advantage of the measurement apparatus is its capacity to be adapted to a wide variety of liquid media, and in particular to continuous cultures of all types, such as for example chemostat, turbidostat or cyclic cultures.
The light source and/or the detection means are preferably designed to measure the intensity of the modified beam around at least one wavelength, the optical property or properties of the medium each corresponding to a wavelength or wavelengths.
In a first preferred embodiment, the optical property or properties of the medium comprise(s) the turbidity of the medium.
The detection means are thus preferably arranged in the direction of the transmitted beam, the received intensity/transmitted intensity ratio being optimal. In an alternative of the embodiment, at least some of the detection means are arranged at an angle to the direction of the transmitted light, so as to detect the diffused light.
In the first embodiment, the light source and/or the detection means are preferably designed for infrared measurements, advantageously around 880 nm.
It is for example advantageous that the light source is a luminescent diode or LED, transmitting in the infrared.
The transmission of infrared light with wavelengths varying from 840 to 920 nm with a peak at 880 nm simplifies the use of the measurement apparatus and gives good accuracy. Since the diffusion of the light depends on the wavelength, the use of white light, for example, makes the apparatus more difficult to use and gives less accurate results than an almost monochromatic infrared light. In addition, the infrared radiation passes more easily through the matter, which is favourable for the measurement of high turbidities. Transmission at other wavelengths or of white light is however also possible.
In a second preferred embodiment, the optical property or properties of the medium comprise(s) the absorbance of the medium, the invention thus applying to the colorimetry and the spectrophotometry of a liquid or solid medium.
In a third preferred embodiment, the optical property or properties of the medium comprise(s) the fluorescence of the medium.
Thus, the GFP (Green Fluorescence Protein) naturally present in the Aequora victoria jellyfish, when excited by UV or blue light (2 excitation peaks at 395 and 475 nm), transmits green light at different wavelengths (509 and 540 nm) by fluorescence, which allows distinction between the incident and diffused light. The transmission does not require the presence of a substrate or cofactor. There are different versions of the gene coding for this protein, which has the effect of improving its quantum yield and modifying its emission spectrum (HELM, R., Nature, vol. 373, pp. 663-664, 1995).
Similarly, it is possible to quantify the intracellular or extracellular compounds transmitting by fluorescence. For example:
Thus the oxido-reduction Coenzyme NADH transmits a fluorescence at 450 nm when it is excited by light at wavelength 350 nm (the oxidised form NAD does not fluoresce). During cell growth, the quantity of NADH varies with the cell concentration (Li, J., Biotechnology and Bioengineering, vol. 37, pp. 1043-1049, 1991).
The same is true for different fluorescent colorants and chromophores (also called fluorochromes or fluorophores), such as fluorescein, fluorescent antibodies, pigments, quinine, etc.
The introduction into different cells (prokaryotes or eukaryotes) of genes coding for GFP and their expression under different conditions thus allows the fluorescence to be continuously monitored. The measurement apparatus according to the invention may also be used to study the internal filter effects of fluorescent media.
This third embodiment has applications in many fields, such as the quantification of fluorescent substances in gels, blots or microplates. In addition, in vivo studies are possible, such as the measurement of gene expression by monitoring the fluorescence of tagged proteins for which they code, leading to both the quantification and cellular localisation of these proteins. These measurements performed in bioreactors can also be used to carry out analyses as a function of time, the physiological state of the cells, and the environmental conditions of the culture.
The measurement apparatus preferably comprises a processing unit linked to the reading means and having storage means, the storage means being designed to store at least one equation determined by a calibration curve giving the optical property or properties of the medium as a function of the intensity of the transmitted light beam for respectively at least one value of the intensity of the modified beam. The processing unit then calculates the optical property or properties of the medium from the stabilised intensity of the transmitted light beam for one of the values of the nominal intensity, on the one hand, and the equation corresponding to this value of the nominal intensity, on the other.
The determination of the turbidity, the absorbance and/or the fluorescence of the medium from these measurements is thus automated, and may be performed in real time.
It is thus advantageous that the processing unit comprises a central processing unit and at least one card for analog/digital and digital/analog conversion, coupling the central processing unit to the control means and the regulation means.
Such cards make current/voltage and voltage/current converter stages unnecessary.
In addition, the processing unit preferably calculates the optical property of the medium directly from a value proportional to the intensity of the transmitted beam.
This embodiment contrasts with known techniques, in which the optical properties are obtained from feedback signals, which must pass through an integration system to be processed. It thus avoids the introduction of inaccuracies in the results.
Advantageously, the measurement apparatus comprises a clock, recording means in the means of storage of representative values of the optical property or properties of the medium, and means of repeated activation of the feedback system and the recording means.
The recording of the results is thus automated, the measurements being advantageously performed at regular intervals so that the variation of the optical property as a function of time can be monitored. In a first embodiment, the values recorded are those directly obtained by reading the intensity of the transmitted beam, these results being processed subsequently. In a second embodiment, the measurement apparatus determines the optical properties after each measurement, and it is these which are recorded in the storage means.
The feedback regulating system and the control means are such that the equalisation of the measured intensity and the nominal intensity of the modified beam is obtained continuously.
Thus, the response time is negligible and only limited by the intrinsic response times of the electronic components, of the order of nanoseconds. This embodiment contrasts with known techniques, in which the adjustment is made by successive trials and governed by periodic impulses. The measurement apparatus can thus offer a high speed of measurement, which can be applied for example in the serial analysis of a very large number of samples and to perform measurements in media which are heterogeneous or perturbed by gas bubble circulation. In these latter cases, the measurement apparatus is equipped with means for performing measurements in bursts, and for carrying out statistical analysis of values collected from each of these bursts, so as to calculate significant values.
A further object of the invention is a micro-bioreactor, equipped with a measurement apparatus according to the invention.
The fact that the measurement apparatus can be completely outside the micro-bioreactor, that it does not require sample removals and corresponding dilutions, and that it enables automation of measurements, enables the construction of a micro-bioreactor with considerable advantages. In particular, a small volume of the culture is sufficient for measurements, which improves the ease of use. The small size and the low frequency of changes of supply vessels for the culture media are other advantages. In addition, the micro-bioreactor and its systems for supplies and removal of effluents may be completely isolated from the external media. Their pressurisation ensures simplicity and reliability, since the use of pipes with known head-loss gives precise flow rates and avoids costly and bulky pumps. This guarantees sterility by preventing any contamination of the supply vessels by the culture and of the culture by the external environment, and allows for example the culture of pathogenic strains in complete safety.
The automation of the measurements also makes possible the automation of sampling for optical densities corresponding to exact physiological states, such as for example maximum growth rate, end of growth or end of stationary phase. It also enables the execution of automatic dilutions for cyclic cultures.
Another advantage is that several micro-bioreactors may be linked in a battery, independently or in cascade, each containing a culture in well-defined and reproducible conditions. The measurement apparatus linked to the different micro-bioreactors are then advantageously linked to a central system designed to control the measurements in parallel.
In a preferred embodiment, the micro-bioreactor comprises:
a tube closed by a stopper, intended to contain a culture,
a first pipe, to inject sterile air and the culture medium into the culture, and
a second pipe, to remove the effluents, the tube and all the pipes being under pressure.
In an advantageous embodiment for cyclic cultures, the micro-bioreactor comprises a dilution system designed to dilute a culture. The measurement apparatus thus comprises a clock and a processing unit linked to the dilution system, the processing unit cyclically triggering the dilution system.
This device is very advantageous for developing strains or for performing physiological studies on the growth parameters such as, for example, the level and yield of growth or measurement of the latency time before the resumption of growth.
The processing unit preferably repeatedly activates the feedback system, records the representative values of the optical property or properties of the medium in the storage means, and triggers the dilution system when it observes a stagnation of the optical property or properties of the medium over a plurality of the latest recorded values.
In alternative embodiments, the processing unit triggers other actions on the medium, such as for example the supply of various solutions or gases, when it observes stagnation.
In another embodiment, the processing unit periodically triggers the dilution system, after a predetermined culture period.
The invention also relates to any system equipped with a measurement apparatus according to the invention, such as for example a bioreactor (adaptation by increasing or internal), an independent apparatus or a microplate reader.
Different types of cultures may also be used: discontinuous (batch) cultures, optionally with continuous or sequential addition of one or more substrates (fed batch); cyclic cultures; turbidostat, enabling the maintenance of a culture turbidity at a constant value by addition of a constant volume of the culture medium into the bioreactor, so that the turbidity oscillates around a reference value; or chemostat, enabling the continuous addition of a constant volume of the culture medium into the bioreactor at a rate lower than the maximum growth rate of the micro-organism.
Various methods of introduction of fluids may be used, such as for example pumps, all-or-nothing or proportional solenoid valves, flowmeters, several supply vessels for different substrates or compounds, added as a function of time or of the optical density and/or several gases.
The pipes are preferably connected by means of connectors each comprising a male part connected to a first pipe, a female part connected to a second pipe, and means of connection of the male part and the female part, each of these male and female parts comprising:
a tapered and hollow end connected to the pipe corresponding to this part and communicating with this pipe, and
a tapered sleeve, surrounding this end and connected to this pipe.
The end of the male part is introduced into the end of the female part, the sleeve of one of the parts is introduced into the sleeve of the other part and the sleeves are fixed to each other by connection means.
The sleeves thus serve to protect the ends, through which the fluid circulates.
The connectors are preferably provided with stoppers to give a sealing closure, and are sterilised with steam, in an autoclave for example. During the connection, the stoppers are removed and the ends of the sleeves are briefly passed over a flame to ensure, their sterility and that of the air in close proximity. This system enables the internal areas of the male and female parts to be kept cold and also permits immediate connection without deterioration of the seals, while retaining complete sterility.
The sealing of the connection between the two ends is advantageously obtained by means of seals attached to the male and female parts respectively and against which the ends of the female and male parts respectively are engaged.
The sleeve of the male part is advantageously introduced into the sleeve of the female part. The protection given by the sleeves is thus still more reliable.
In addition, it is advantageous that the connection means comprise a locking ring linked to the pipe corresponding to the receptor sleeve and a fixing system (for example a screw thread) on the sleeve introduced into the receptor sleeve and engaging this ring.
The locking ring is advantageously machined to include a stop limiting the locking. This embodiment avoids excessive compression of the seals which would cause their deterioration.
In another embodiment, O-ring sealing rings are added to the internal areas of the male and female parts. They reinforce the safety and sealing.
The invention also relates to a connector, and to the male and female parts of such a connector, particularly suited to the micro-bioreactor of the invention and corresponding to the above description.
A further object of the invention is a method for measuring at least one optical property of a medium, in which:
a light beam of variable intensity is transmitted towards or into the medium,
the intensity of the beam modified by the medium is measured,
the intensity of the transmitted beam is adjusted so that the intensity of the modified beam is equal to a fixed nominal value,
the stabilised intensity of the transmitted beam thus obtained is read, and
a value representative of the stabilised intensity is related to an equation determined by a curve giving the optical property or properties as a function of the intensity of the representative value of the stabalised intensity of the transmitted beam for the nominal intensity of the modified beam, so as to calculate the optical property or properties of the medium.
According to the invention, the nominal intensity is adjusted so that the stabilised intensity of the transmitted beam is within a predetermined range.